Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for eight weeks using a supplemented medium 199 (199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions and desmosomes), a brush border on the apical surface, and keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by co-cultivation with either lethally-irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, "floating" substrates compared with substrates attached directly to the bottom of the culture dish.